This invention relates generally to the cloning and identification of novel outer membrane proteins of several strains of Neisseria, and more specifically to proteins useful in the prevention, therapy and/or diagnosis of infection and diseases in mammals caused by these strains.
The pathogenic Neisseriae cause several important non-symptomatic infections and symptomatic diseases in humans. Neisseria gonorrhoeae is the agent of non-symptomatic gonococcal infection or symptomatic disease, i.e., gonorrhea. Neisseria meningitidis is the agent of a rapidly progressive spinal meningitis, which may also have a non-symptomatic infective stage. The surfaces of such pathogens provide crucial interfaces for interactions between the pathogen and the host. Many bacterial virulence factors are outer membrane proteins, and surface exposed proteins are the primary targets recognized and attacked by the host""s immune system. Thus, the role of outer membrane proteins is of particular importance in understanding the pathogenesis of these organisms. The most abundant and immunodominant outer membrane proteins of the pathogenic Neisseriae have been studied extensively (Sparling P. F. et al, Clin. Invest., 89: 1699-1705 (1992)). For example, it is known that the immunodominant components of the gonococcal surface are antigenically variant, suggesting that this organism is capable of adapting to varying host environments while avoiding host immune responses. Although the major gonococcal surface proteins have been extensively studied, little is known about less abundant proteins and their contributions to pathogenesis.
Two-dimensional electrophoresis (IEF and SDS-PAGE) of labeled, e.g., radioiodinated or biotinylated, gonococcal surface proteins suggested that numerous ( greater than 20) of the less abundant gonococcal outer membrane proteins remained uncharacterized (unpublished observations). Among these might be proteins which play an important role in infection.
For example, surface-exposed outer membrane proteins of other microorganisms, e.g., Haemophilus influenzae D15 surface antigen (D-15-Ag) and the Pasteurella multocida Oma87, have been found to be useful in eliciting antibodies that were protective against infectious challenge in animal models. The Omp85-like D-15-Ag was conserved in both non-typeable and typeable strains of H. influenzae and was recognized by convalescent patient sera; affinity-purified anti-D-15-Ag serum was protective in the rat pup model (Thomas, W. R., et al, Infect. Immun., 58:1909-1913 (1990); Flack, F. S. et al, Gene, 156:97-99 (1995)). H. influenzae serotypes a-f, nontypeable H. influenzae and Haemophilus parainfluenzae all expressed proteins similar to the D-15-Ag, as demonstrated by immunoblot analysis. Antibodies to recombinant D-15-Ag protected against H. influenzae type b and type a bacteremia in the infant rat model (Loosmore, S. M. et al, Infect. Immun., 65:3701-3707 (1997)).
Like H. influenzae D-15-Ag, the Oma87 of P. multocida was highly conserved among strains and was recognized by protective antibody; it was present in all 16 serotypes of P. multocida and was recognized by convalescent animal sera. Antibodies raised to recombinant Oma87 were protective against homologous challenge in the mouse model (Ruffolo, C. G. et al., Infect. Immun., 64:3161-3167 (1996)). Despite the several publications describing the immunological properties of D-15-Ag and Oma87, the function of these proteins remains unknown.
There remains a need in the art for the development of proteins from Neisseriae which are useful in research, diagnosis and treatment of the infections, especially non-symptomatic infections, and the diseases caused by these pathogens.
In one aspect, the invention provides an isolated outer membrane protein of N. gonorrhoeae having an apparent molecular weight of 85 kDa and characterized by an amino acid sequence of SEQ ID NO: 2, a fragment, an analog or a homolog thereof.
In another aspect, the invention provides a nucleic acid sequence encoding the Omp85 of N. gonorrhoeae or a fragment thereof.
In still another aspect, the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the Omp85 of N. gonorrhoeae or a fragment thereof under the control of suitable regulatory sequences which direct expression of said Omp85 protein or fragment in a selected host cell.
In yet a further aspect, the invention provides a host cell transformed with the above described nucleic acid molecule.
In still a further aspect, the invention provides a method of recombinantly expressing the Omp85 of N. gonorrhoeae or a fragment thereof comprising the steps of culturing a recombinant host cell transformed with a nucleic acid sequence encoding said protein or fragment under conditions which permit expression of said protein or peptide.
In another aspect, the invention provides a method for preparing an Omp85 protein of N. gonorrhoeae or fragment thereof comprising chemically synthesizing said protein or fragment.
In yet another aspect, the invention provides a diagnostic reagent comprising a nucleic acid sequence encoding Omp85 of N. gonorrhoeae, isolated from cellular materials with which it is naturally associated, a sequence complementary thereto, a fragment thereof, a sequence which hybridizes thereto under stringent conditions, an allelic variant thereof, a mutant thereof, or a sequence encoding Omp85 or a fragment thereof fused to a sequence encoding a second protein, and a detectable label which is associated with said sequence.
In still another aspect, the invention provides an isolated antibody which is directed against Omp85 of N. gonorrhoeae or a fragment thereof.
In a further aspect, the invention provides an anti-idiotype antibody specific for the antibody described above.
In another aspect, the invention provides a diagnostic reagent comprising the antibody or anti-idiotype antibody described above and a detectable label.
In yet another aspect, the invention provides a vaccine composition comprising an effective amount of a Omp85 protein of N. gonorrhoeae, a fusion protein or fragment thereof and a pharmaceutically acceptable carrier. This composition can also include at least one other antigen or fragment thereof.
In another aspect, the invention provides a vaccine composition comprising an effective amount of a nucleic acid sequence encoding the Omp85 protein of N. gonorrhoeae, a fusion protein, or a fragment thereof and a suitable nucleic acid delivery vehicle. This vaccine composition may also be polyvalent.
In still a further aspect, the invention provides a method of vaccinating a human or animal against gonococcal infection or disease comprising administering to said human or animal a composition comprising an effective amount of at least one of the compositions described above.
Another aspect of the present invention includes a method for diagnosing gonococcal infection or disease in a human or animal comprising the steps of contacting an Omp85 antigen optionally associated with a detectable label or a homolog thereof with a biological sample from a human subject to be diagnosed, wherein the presence of naturally occurring antibodies to N. gonorrhoeae in said sample permits the formation of an antigen-antibody complex, and analyzing said sample for the presence of said complex, which indicates infection with N. gonorrhoeae. 
Still another aspect of the invention provides a method for diagnosing gonococcal infection or disease in a human or animal comprising the steps of: contacting an Omp85 antibody, optionally associated with a detectable label, with a biological sample from a human subject to be diagnosed, wherein the presence of naturally occurring N. gonorrhoeae Omp85 in said sample permits the formation of an antigen-antibody complex, and analyzing said sample for the presence of said complex, which indicates infection with N. gonorrhoeae. 
Yet a further aspect of the invention provides a method for diagnosing gonococcal infection or disease in a human or animal comprising the steps of: employing a nucleic acid sequence encoding all or a portion of an Omp85 antigen or an Omp85 antibody, optionally associated with a detectable label, as a probe which, when in contact with a biological sample from a human subject to be diagnosed, enables detection of infection by hybridization or amplification of nucleic acid sequences of N. gonorrhoeae Omp85 in said sample.
Yet a further aspect of the invention includes a therapeutic composition useful in treating humans or animals with non-symptomatic gonococcal infection or symptomatic disease comprising at least one anti-N. gonorrhoeae Omp85 antibody and a suitable pharmaceutical carrier.
In still another aspect, the invention includes a method for treating non-symptomatic gonococcal infection or symptomatic disease in a mammalian host comprising administering an effective amount of the therapeutic composition described above.
In yet another aspect, the invention provides a kit for diagnosing infection with N. gonorrhoeae in a human or animal comprising an Omp85 protein or fragment thereof or an anti-Omp85 antibody or a nucleic acid sequence encoding the protein or antibody as described above.
In another aspect, the invention provides a method of identifying compounds which specifically bind to Omp85 of N. gonorrhoeae or a fragment thereof, comprising the steps of contacting said Omp85 protein or fragment with a test compound to permit binding of the test compound to Omp85; and determining the amount of test compound which is bound to Omp85.
In still another aspect, the invention provides a compound identified by the above method.
In one aspect, the invention provides an isolated outer membrane protein of N. meningitidis having an apparent molecular weight of 85 kDa and characterized by an amino acid sequence of SEQ ID NO: 4, a fragment, an analog or a homolog thereof.
In another aspect, the invention provides a nucleic acid sequence encoding the Omp85 of N. meningitidis or a fragment thereof.
In still another aspect, the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the Omp85 of N. meningitidis or a fragment thereof under the control of suitable regulatory sequences which direct expression of said Omp 85 or fragment in a selected host cell.
In yet a further aspect, the invention provides a host cell transformed with the above described nucleic acid molecule.
In still a further aspect, the invention provides a method of recombinantly expressing the Omp85 of N. meningitidis or a fragment thereof comprising the steps of culturing a recombinant host cell transformed with a nucleic acid sequence encoding said protein or fragment under conditions which permit expression of said protein or peptide.
In another aspect, the invention provides a method for preparing an Omp85 protein of N. meningitidis or fragment thereof comprising chemically synthesizing said protein or fragment.
In yet another aspect, the invention provides a diagnostic reagent comprising a nucleic acid sequence encoding Omp85 of N. meningitidis, isolated from cellular materials with which it is naturally associated, a sequence complementary thereto, a fragment thereof, a sequence which hybridizes thereto under stringent conditions, an allelic variant thereof, a mutant thereof, or a sequence encoding Omp85 or a fragment thereof fused to a sequence encoding a second protein, and a detectable label which is associated with said sequence.
In still another aspect, the invention provides an isolated antibody which is directed against Omp85 of N. meningitidis or a fragment thereof.
In a further aspect, the invention provides an anti-idiotype antibody specific for the antibody described above.
In another aspect, the invention provides a diagnostic reagent comprising the antibody or anti-idiotype antibody described above and a detectable label.
In yet another aspect, the invention provides a vaccine composition comprising an effective amount of a Omp85 protein of N. meningitidis, a fusion protein or fragment thereof and a pharmaceutically acceptable carrier. This composition can also include at least one other antigen or fragment thereof.
In another aspect, the invention provides a vaccine composition comprising an effective amount of a nucleic acid sequence encoding the Omp85 protein of N. meningitidis, a fusion protein, or a fragment thereof and a suitable nucleic acid delivery vehicle. This vaccine composition may also be polyvalent.
In still a further aspect, the invention provides a method of vaccinating a human or animal against non-symptomatic meningococcal infection and symptomatic disease comprising administering to said human or animal a composition comprising an effective amount of at least one of the compositions described above in either a pharmaceutically acceptable carrier or a nucleic acid delivery system.
Another aspect of the present invention includes a method for diagnosing non-symptomatic gonococcal infection or symptomatic disease in a human or animal comprising the steps of contacting an Omp85 antigen optionally associated with a detectable label or a homolog thereof with a biological sample from a human subject to be diagnosed, wherein the presence of naturally occurring antibodies to N. meningitidis in said sample permits the formation of an antigen-antibody complex, and analyzing said sample for the presence of said complex, which indicates infection with N. meningitidis. 
Still another aspect of the invention provides a method for diagnosing non-symptomatic meningococcal infection and symptomatic disease in a human or animal comprising the steps of: contacting an Omp85 antibody, optionally associated with a detectable label, with a biological sample from a human subject to be diagnosed, wherein the presence of naturally occurring N. meningitidis Omp85 in said sample permits the formation of an antigen-antibody complex, and analyzing said sample for the presence of said complex, which indicates infection with N. meningitidis. 
Yet a further aspect of the invention provides a method for diagnosing non-symptomatic meningococcal infection and symptomatic disease in a human or animal comprising the steps of: employing a nucleic acid sequence encoding all or a portion of an Omp85 antigen or an Omp85 antibody, optionally associated with a detectable label, as a probe which, when in contact with a biological sample from a human subject to be diagnosed, enables detection of infection by hybridization or amplification of nucleic acid sequences of N. meningitidis Omp85 in said sample.
Yet a further aspect of the invention includes a therapeutic composition useful in treating humans or animals with non-symptomatic meningococcal infection and symptomatic disease comprising at least one anti-N. meningitidis Omp85 antibody and a suitable pharmaceutical carrier.
In still another aspect, the invention includes a method for treating non-symptomatic meningococcal infection and symptomatic disease in a mammalian host comprising administering an effective amount of the therapeutic composition described above.
In yet another aspect, the invention provides a kit for diagnosing infection with N. meningitidis in a human or animal comprising an Omp85 protein or fragment thereof or an anti-Omp85 antibody or a nucleic acid sequence encoding the Omp85 protein or antibody, as described above.
In another aspect, the invention provides a method of identifying compounds which specifically bind to Omp85 of N. meningitidis or a fragment thereof, comprising the steps of contacting said Omp85 protein or fragment with a test compound to permit binding of the test compound to Omp85; and determining the amount of test compound which is bound to Omp85.
In still another aspect, the invention provides a compound identified by the above method.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof, reference being made to the accompanying figures.